Figure S1. Validation of RNF43 mutation c.1389_1392dupCAGT. (A) Sanger sequencing of the c.1389_1392dupCAGT somatic mutation. The 4 bp duplication was identified by Sanger sequencing, although it was difficult to distinguish from the sequencing background, due to the low mutant allele frequency in the tumour. The wild-type (WT) and mutant (Mut) sequences are indicated above the sequencing trace of the tumour and germline samples, with the mutated positions indicated by the asterisks. Positions where the mutant allele can be seen in the tumour at a low frequency are indicated by the arrows. (B) Allele-specific PCR for validation of the c.1389_1392dupCAGT mutation. Wild-type and mutant-specific primers were used to amplify the tumour and matched germline samples. Reactions were separated and visualized by gel electrophoresis, along with a 50 bp ladder to estimate fragment size. Amplification of both reactions in the tumour indicates the presence of both wild-type and mutant alleles in this sample, while only wild-type alleles are present in the matched germline control, confirming the somatic status of the mutation. (C) Molecular cloning of the c.1389_1392dupCAGT mutation. Six of 50 mutation-positive clones were identified by sequencing the extracted DNA from picked colonies. Sequence traces for two wild-type clones and the matched germline control are also shown.

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